This invention relates to an assay for an analyte, and more particularly to a solid phase assay. In a solid phase assay, a binder specific for at least the ligand to be determined (analyte) is supported on a solid support, whereby, in the assay it is not necessary to employ an additional agent for separating the bound and free phases formed in the assay.
There are known in the art assays for analytes wherein the tracer employed in the assays includes a particular metal label, such as, a colloidal metal particle. Thus, for example, in such an assay, a binder specific for the analyte is supported on a solid support, and the tracer is comprised of a ligand specific for the analyte, which ligand of the tracer is labeled with a particular colloidal metal label. In such an assay, the tracer is indirectly attached to the binder on the solid support by binding of the analyte to the binder and binding of the tracer to the analyte, whereby the presence and/or amount of the analyte in a sample can be determined by detecting the presence and/or amount of colloidal metal tracer, which is indirectly bound to the binder on the solid support. A number of such assays are described in the European Patent Publication Nos. 158,746 and 293,947.
The signal generated by such a tracer can significantly be improved by subjecting the metal label indirectly bound to the surface of the solid phase to a so-called physical developing procedure.
The art-known physical developers generally consist of a solution containing a soluble metal salt, such as silver nitrate, a reducing agent, such as hydroquinone and an appropriate buffer system to establish a specific pH.
The European Patent Publication No. 158,746, page 10 lines 18 to 32, describes a method to improve the signal of a colloidal metal label significantly by subjecting the metal label indirectly bound to the surface of a blotting medium to a so-called physical developing procedure.
In case a silver based physical developer is applied the reduction of silver ions to metallic silver is catalyzed at the surface of the metal label, resulting in a specific deposition of metallic silver at the metal label site. In turn, the thus formed metallic silver particles catalyze the reduction, creating an auto-catalytic process. The effect of a physical development is that the reddish optical gold signal turns into a deep-brown to black silver signal, with a much higher intensity. The use of this art-known physical developer technique thus results in an improved signal, although there are a number of drawbacks associated with it.
One of the major problems in the use of physical developers is the solubility of the metal salts. Indeed, it is well known that metal ions, such as silver ions, form insoluble salts with many counter ions. Apart from depleting the available silver ion supply, these insoluble salts also form nuclei at which the reduction process is catalyzed as well, which results in a seriously augmented noise level. Moreover, silver ions may form light sensitive silver salts, such as silver bromide and silver chloride, which are readily reduced to metallic silver under the influence of light, starting an auto-catalytic process. It is therefore absolutely necessary to work with extremely clean contacting surfaces, e.g. vessels, analytical grade chemicals and ultra-pure water. Usually it is also necessary to introduce multiple washing steps between the incubation with the metal tracer and the physical development of the label in order to remove unwanted ions present in the incubation medium. All this tends to make traditional physically developed metal-based assays more complex, expensive and error prone.
The major disadvantage of the traditional methods lies within the nature of physical developing itself. In the case of a silver-based physical developer, for example, the reducing agent reduces all silver ions at a certain rate. To obtain optimal sensitivity the amplification process has to be aborted by removing the physical developer from the metal label-containing phase before the non-label-induced reduction, the `so-called selfnucleation`, becomes apparent. It is obvious that physical developers become more flexible and powerful if the ratio between metal-specific reduction and self-nucleation can be increased. With the traditionally used assays, this ratio can hardly be augmented. The only parameter which can be modulated is the overall speed of the process; selfnucleation can be postponed only at the expense of a slower metal label amplification. One of the most obvious ways to do this is to change the concentration, nature or environment of the reducing agent. A frequently used approach is the use of hydroquinone at a pH lower than 4. The reducing action of hydroquinone is strongly inhibited in an acid environment; the user of the physical developer therefore has enough time to stop the metal label amplification before self-nucleation causes too much noise. However, acid additions are in many cases not compatible with the nature of the binding between the marked specific binding agent and its corresponding bindable substance. Most monoclonal antibodies have only a low or average affinity to their antigens at said pH. Moreover, no real gain in sensitivity can be accomplished because the label amplification is slowed down to the same degree as the self-nucleation is slowed down.
Thus there is a strong need for improving the sensitivity and practicality of metal based detection solid phase assays. Surprisingly it has been found that a severalfold increase in the ratio between the tracer bound signal and the self-nucleation can be obtained using the device described hereinafter, thus resulting in a higher sensitivity and speed.